Project Summary/Abstract Hepatitis C virus (HCV) infects approximately 3% of the world's population, often causing associated diseases including cirrhosis of the liver and hepatocellular carcinoma (HCC). With limited effective therapies and slow vaccine development, alternative approaches for treatment and prevention of infection and its associated diseases is imperative. An immune-based approach for targeted therapy utilizes adoptive T cell transfer to genetically engineer an individual's T cells to become reactive to an antigen of choice. Our lab has previously demonstrated its ability to successfully redirect peripheral blood lymphocyte (PBL)-derived T cells with retroviral vectors in order to recognize target antigens. Studies redirecting T cells to recognize melanoma- associated antigens have led to ongoing Phase I/II clinical trials for treating metastatic disease. We believe this approach can be applied to HCV infections and HCV-associated malignancies. Our lab has cloned and expressed two functional TCRs from PBL-derived T cell clones from humans capable of recognizing HCV peptides NS3:1406-1415 and NS3:1073-1081. Additionally, we have shown that these are high affinity TCRs since they have CD8-independent target cell recognition and can recognize naturally occurring mutant escape variants. We believe the approach to redirect T cells to recognize mutagenic HCV antigens may have benefits in understanding TCR biology as well as producing a clinical therapy for HCV infection and associated diseases. Based on these observations, we hypothesize that the ability of a TCR to have broad reactivity is not unique to such isolated clones and that in patients with resolved HCV infection there may exist a variety of TCRs with the capability of recognizing many epitopes of viral proteins. We also hypothesize that HCV TCR transduced T cells have the ability to recognize and kill tumor cells expressing wild type and mutant HCV antigens in vitro and can mediate regression of HCV+ tumor cells in vivo. Three specific aims have been developed to examine these hypotheses. Aim 1 will test our hypothesis that HCV reactive T cells from patients who resolved their HCV infections but not patients with HCV chronic infections express TCRs that recognize wild type and a broad spectrum of mutant HCV antigens. Aim 2 will test the hypothesis that the TCRs from HCV reactive T cells from patients who resolved their HCV infections can transfer this reactivity against mutant HCV antigens to other T cells. Aim 3 will test the hypothesis that HCV TCR transduced T cells can mediate regression of HCC tumors expressing wild type and mutant HCV antigens in mouse xenograft tumor models in vivo. Overall, this proposal will yield novel information that may lead to a better understanding of the capability of TCRs to recognize multiple mutant HCV epitopes and will aid in the development of novel therapeutics for patients with HCV-associated HCC.